Project Summary We have recently determined that ATP is released from the urinary bladder epithelium (urothelium) in response to stretch or chemical stimulation through pannexin channels. However, we have also found that pannexin channel blockers only show a minor effect on increased urothelial ATP release in response to stimulation of Toll-like receptors (TLRs) by bacterial endotoxins. This raises the possibility that ATP is released from the urothelium by at least two distinct mechanisms; one for physiological stimuli such as stretch, and one for pathological stimuli such as infection. In this application, we propose to examine the mechanism of the endotoxin-mediated ATP release, in the hopes that it will give us better insights into the etiology of pathological bladder disorders. Through a collaborative project with Dr. Claire Mitchell?s group at the University of Pennsylvania, we have discovered that stimulation of TLRs in various cell types of the eye can cause the release of ATP stores through lysosomal exocytosis. Stimulation of retinal epithelial cells or optic nerve astrocytes with TLR agonists releases both ATP and lysosomal enzymes, which can be blocked by glycyl-L-phenylalanine 2-naphthylamide (GPN), a drug that destroys lysosomes. We believe that this released ATP can then act on nearby purinergic receptors to induce the activation of the inflammasome and ultimately release a number of pro-inflammatory cytokines. Thus, lysosomal ATP release may represent an important step in the induction of inflammation in a number of cell types. In our current project, we propose to determine if lysosomal exocytosis is a mechanism of ATP release from the urothelium in response to TLR stimulation. To accomplish this we will demonstrate that LPS- stimulation of primary rat urothelial cultures releases ATP as well as lysosomal enzymes, such as acid phosphatase and that this release is attenuated following lysosomal disruption with GPN. We will also measure, using flow cytometry, the presence of the lysosomal marker LAMP1 on the cell surface, which would indicate lysosomal fusion with the plasma membrane. In Aim #2, we will demonstrate a role for lysosomal ATP release in bladder pathology, by showing a decrease in LPS-mediated excitation of bladder reflexes and inflammation after lysosomal disruption with GPN. It is our hope that these experiments will clearly indicate that lysosomal ATP release plays a significant role in LPS-induced bladder inflammation and hyperactivity, confirming the importance of urothelial lysosomes in the etiology of pathological bladder disorders.